Amino-acid based radicals are involved in a range of productive and destructive processes in living organisms. Functional, and typically highly controlled, radical chemistry occurs in enzymes that use amino acids as catalytically active redox cofactors. In contrast, oxidative stress conditions are well known to generate a range of uncontrolled, and for the organism, potentially harmful protein radical reactions. Despite the fact that the number of known amino-acid radical enzymes and pathological conditions linked to dysfunctional amino-acid radical chemistry continuously increases, experimental characterization of basic thermodynamic parameters (i.e. reduction potentials and pKA values) involved in protein radical formation, stabilization, and long-range migration is still rudimentary. This situation reflects the simple fact that the characteristically reactive and thermodynamically hot radical state is highly challenging to study in the natural systems. The focus of my Ph.D. research project is to extend and explore a library of well-structured model proteins specifically made to study protein radical chemistry. These model proteins contain the radical site of interest as well as features that will facilitate the biophysical characterization of the radical and its surounding protein environment. Using these model systems, we aim to systematically map the thermodynamic properties of amino-acid radicals as a function of protein structural features. The overall objective with the proposed experimental studies is to correlate specific structural features with the reduction potentials and pKA values of tyrosine radicals. These properties will be studied as a function of electrostatic interactions, hydrogen-bonding interactions, degree of solvent exposure and redox-coupled protonic reactions within the protein scaffold. Experimentaly, the two main methods that will be utilized are pulsed voltammetry techniques for electrochemical analyses and high- resolution protein NMR spectroscopy for structural studies. Specific Aim 1 includes work to probe radical cofactor-solvent interactions and the influence of these interactions on the redox properties of tyrosine. Specific Aim 2 involves the characterization of tyrosine radical induced Bohr effects i.e. redox-coupled acid/base reactions within the protein scaffold. Finally, Specific Aim 3 involves efforts to extend our family of radical model proteins to include model proteins of natural origin, such as ubiquitin. The obtained data will provide an essential and unique link between the natural systems, small-molecule solution studies and theoretical work. The proposed studies have a high potential to provide data that are directly relevant to thermodynamic discussions of natural amino-acid radical systems and evaluations of mechanistic models. The broad, more long-term objective with the proposed studies is to build a platform on which to analyze the properties of functional and dysfunctional amino-acid radicals in natural systems.